Polypeptide Containing DNA-Binding Domain

ABSTRACT

The present invention provides an artificial nuclease comprising a DNA-binding domain and a function domain linked to each other via a polypeptide consisting of 35 to 55 amino acid residues wherein amino acid residues at two sites in a DNA-binding module contained in a DNA-binding domain exhibit a mode of repetition that is different for every four DNA-binding modules; a vector for expressing said artificial nuclease; a vector library for preparing said vector; and a vector set for preparing said vector library.

A computer readable text file, entitled “SequenceListing.txt,” created on or about Jan. 28, 2016 with a file size of about 41 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a polypeptide containing a DNA-binding domain and a function domain. The present invention also relates to a vector comprising a polynucleotide coding for said polypeptide. The present invention further relates to a vector library for preparing said vector. The present invention also relates to a vector set for preparing said vector library. The present application claims the priority based on Japanese patent application No. 2013-166768 filed on Aug. 9, 2013, a whole content of which is incorporated herein by reference.

BACKGROUND ART

For a polypeptide comprising a plurality of nuclease subunits consisting of a DNA-binding domain and a function domain, TALEN (TALE Nuclease), ZFN (Zinc Finger Nuclease) and the like are known (Patent references 1-4, Non-patent references 1-5). For instance, an artificial nuclease is known in which a DNA-cleaving domain is used as a function domain. These artificial nucleases cause cleavage of DNA duplicates by a multimer formed by a plurality of DNA-cleaving domains approaching close together around a binding site of a DNA-binding domain. A DNA-binding domain comprises repetition of plurality of DNA-binding modules and the respective DNA-binding module recognizes a specific base pair in a DNA strand. Thus, by suitably designing DNA-binding modules, it becomes possible to specifically cleave a sequence of interest. By utilizing errors and recombinations that occur when sequence specific cleavage is subject to repair, it is possible to introduce deletion, insertion and mutation of a gene on a genomic DNA. Therefore, these nucleases may be applied for various genetic modifications such as a genome editing (cf. Non-patent reference 6).

Non-patent reference 1 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. Non-patent reference 1 discloses a nuclease in which a single amino acid residue in a specific site in a DNA-binding module other than a DNA recognition site is periodically varied for the every four DNA-binding modules used. However, Non-patent reference 1 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA binding domain. Non-patent reference 1 also fails to disclose that a plurality of amino acid resides in a DNA-binding module are periodically varied. Non-patent reference 1 also fails to disclose the significance and the purpose of the periodical variation.

Non-patent reference 2 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 63 amino acid residues. Non-patent reference 2 also discloses a polypeptide in which amino acid residue(s) in a DNA-binding module other than those amino acid residues in a DNA recognition site is/are periodically varied for the every four DNA-binding modules used. However, Non-patent reference 2 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Non-patent reference 2 also does not refer to anything about the purpose and the significance of the periodical variation for every four DNA-binding modules.

Non-patent reference 3 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. Non-patent reference 3 also discloses a polypeptide in which amino acid residue(s) in a DNA-binding module other than those amino acid residues in a DNA recognition site is/are varied at random for the every DNA-binding modules used. However, Non-patent reference 3 fails to disclose that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Non-patent reference 3 also fails to disclose the purpose and the significance of the variation at random. Non-patent reference 3 also fails to disclose the periodical variation for every DNA-binding module.

Non-patent reference 4 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 63 amino acid residues. Non-patent reference 5 discloses a polypeptide in which a DNA-binding domain and a function domain are linked to each other via a linker of 47 amino acid residues. However, neither Non-patent reference 4 nor Non-patent reference 5 discloses that the used polypeptide has compatibility between a high level of desired function of a function domain and a high level of specificity of sequence recognition of a DNA-binding domain. Also, neither Non-patent reference 4 nor Non-patent reference 5 discloses the periodical variation for every DNA-binding module.

PATENT REFERENCES

Patent reference 1: WO 2011/072246

Patent reference 2: WO 2011/154393

Patent reference 3: WO 2011/159369

Patent reference 4: WO 2012/093833

NON-PATENT REFERENCES

Non-patent reference 1: Nucleic Acids Res. 2011 November; 39(21):9283-93.

Non-patent reference 2: Nat Biotechnol. 2011 Aug. 5; 29(8):697-8.

Non-patent reference 3: Nat Biotechnol. 2011 February; 29(2):143-8.

Non-patent reference 4: Nature. 2012 Nov. 1; 491(7422):114-8.

Non-patent reference 5: Genes Cells. 2013 April; 18(4):315-26.

Non-patent reference 6: Cell. 2011 Jul. 22; 146(2):318-31.

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by the Invention

It is expected that efficiency for obtaining a desired result is improved when a polypeptide comprising a DNA-binding domain with high function of a function domain is used. For instance, in case of an artificial nuclease comprising a DNA-binding domain and a DNA-cleaving domain, it is expected that probability of DNA cleavage is improved to improve efficiency for obtaining cells with a genetic modification of interest when such a nuclease as having a high DNA cleavage activity is used. However, a conventional polypeptide comprising a DNA-binding domain, when showing a high activity of a function domain, is likely to exert a high function also in the region other than a target nucleotide sequence of a DNA-binding domain and thus is not appropriate in view of safety. As such, it was difficult to establish compatibility between a high level of specificity of DNA sequence recognition and a high level of function of a function domain. Besides, cumbersome procedures such as introduction of repetition of DNA-binding modules corresponding to target sequences into a vector are necessary for preparing a polypeptide comprising a DNA-binding domain. Thus, there is a need for a polypeptide that can be prepared with simpler procedures more rapidly.

Therefore, an object of the present invention is to provide a polypeptide, which has compatibility between a high level of function of a function domain and a high level of specificity of DNA sequence recognition, can safely exert desired function with high probability, and can be prepared with simple procedures.

Means for Solving the Problems

The present inventors have earnestly studied to solve the above problems and as a result have found that a polypeptide wherein a DNA-binding domain and a function domain are linked to each other via a polypeptide consisting of 35 to 55 amino acid residues and wherein amino acid residues at two specific sites in a DNA-binding module contained in a DNA-binding domain exhibit a mode of repetition that is different for every four DNA-binding modules has compatibility between a high level of function of a function domain and a high level of specificity of DNA sequence recognition. A vector for expressing said polypeptide could be prepared with simple procedures by using a vector set of specific features and a vector library of specific features.

Thus, in the first aspect, the present invention provides a polypeptide comprising a DNA-binding domain and a function domain,

-   -   wherein the DNA-binding domain and the function domain are         linked to each other via a polypeptide consisting of 35 to 55         amino acid residues,     -   wherein the DNA-binding domain comprises a plurality of         DNA-binding modules consecutively from the N-terminus,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4n-3 counted from the N-terminus is the same for any of         n,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4n-2 counted from the N-terminus is the same for any of         n,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4n-1 counted from the N-terminus is the same for any of         n,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4 n counted from the N-terminus is the same for any of         n,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4n-3 counted from the N-terminus, a combination of an         amino acid residue at position x and an amino acid residue at         position y in a DNA-binding module at position 4n-2 counted from         the N-terminus, a combination of an amino acid residue at         position x and an amino acid residue at position y in a         DNA-binding module at position 4n-1 counted from the N-terminus,         and a combination of an amino acid residue at position x and an         amino acid residue at position y in a DNA-binding module at         position 4 n counted from the N-terminus are different from each         other, and     -   wherein n is natural number of 1 to 10, x is natural number of 1         to 40, y is natural number of 1 to 40, and x and y are different         natural number from each other.

In the second aspect, the present invention provides the polypeptide of the first aspect wherein the function domain is a DNA-cleaving domain.

In the third aspect, the present invention provides a vector comprising a polynucleotide coding for the polypeptide of the first aspect or the second aspect.

In the fourth aspect, the present invention provides a vector library for preparing the vector of the third aspect,

-   -   wherein the vector library consists of a plurality of vectors,         each of which vector has a first restriction site, a         polynucleotide coding for four DNA-binding modules and a second         restriction site in this order from the 5′-end,     -   wherein a combination of the first restriction site and the         second restriction site is any one of a combination of a         restriction site of type A and a restriction site of type B, a         combination of a restriction site of type A and a restriction         site of type C, a combination of a restriction site of type A         and a restriction site of type D, a combination of a restriction         site of type A and a restriction site of type E, a combination         of a restriction site of type B and a restriction site of type         C, a combination of a restriction site of type C and a         restriction site of type D, and a combination of a restriction         site of type D and a restriction site of type E, wherein each of         the restriction sites of types A to E produce different cleaved         terminals when cleaved with the same restriction enzyme; and         among the four DNA-binding modules,     -   wherein, a combination of an amino acid residue at position x         and an amino acid residue at position y in a DNA-binding module         at position 1 counted from the 5′-terminus is the same for any         of the vectors,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 2 counted from the 5′-terminus is the same for any of         the vectors,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 3 counted from the 5′-terminus is the same for any of         the vectors,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 4 counted from the 5′-terminus is the same for any of         the vectors, and     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module at         position 1 counted from the 5′-terminus, a combination of an         amino acid residue at position x and an amino acid residue at         position y in a DNA-binding module at position 2 counted from         the 5′-terminus, a combination of an amino acid residue at         position x and an amino acid residue at position y in a         DNA-binding module at position 3 counted from the 5′-terminus,         and a combination of an amino acid residue at position x and an         amino acid residue at position y in a DNA-binding module at         position 4 counted from the 5′-terminus are different from each         other, and     -   wherein x is natural number of 1 to 40, y is natural number of 1         to 40, and x and y are different natural number from each other.

Furthermore, in the fifth aspect, the present invention provides a vector set for preparing the vector library of the fourth aspect,

-   -   wherein the vector set comprises a plurality of vectors, each of         which vector comprises a first restriction site, a DNA-binding         module and a second restriction site in this order from the         5′-end,     -   wherein the first restriction site and the second restriction         site produce different cleaved terminals when cleaved with the         same restriction enzyme,     -   wherein a combination of an amino acid residue at position x and         an amino acid residue at position y in a DNA-binding module is         any of the four different combinations, and     -   wherein x is natural number of 1 to 40, y is natural number of 1         to 40, and x and y are different natural number from each other.

EFFECTS OF THE INVENTION

The polypeptide of the present invention accomplishes a high level of function of a function domain and simultaneously a high level of specificity of DNA sequence recognition. Thus, by introducing a vector comprising a polynucleotide coding for the polypeptide of the present invention into cells, a desired result can be attained safely with high probability. Besides, by using the vector library of the present invention, a vector for expressing a polypeptide that has compatibility between a high level of function of a function domain and a high level of specificity of DNA sequence recognition can be prepared with simple procedures rapidly. Furthermore, by using the vector set of the present invention, the vector library of the present invention can be prepared with simple procedures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration showing structural features and process for the preparation of the vector set, the vector library and the vector of the present invention.

FIG. 2 shows an embodiment of an amino acid sequence and a nucleotide sequence of a DNA-binding module contained in the vector set, the vector library and the vector of the present invention.

FIG. 3 shows an embodiment of an amino acid sequence and a nucleotide sequence contained in the vector of the present invention.

FIG. 4A shows structural features of the vectors prepared by the methods of Example 3, and Comparative Examples 1 to 3.

FIG. 4B shows structural features of the vectors prepared by the methods of Example 3, and Comparative Examples 1 to 3.

FIG. 4C shows comparison of sequence specificity of nucleases expressed by a combination of various vectors.

FIGS. 5A and 5B show a design of vectors prepared by the methods of Example 3, and Comparative Examples 1 to 3 (FIG. 5A) and comparison of a level of a DNA cleavage activity of nucleases expressed by these vectors (FIG. 5B).

FIGS. 6A and 6B show a design of vectors prepared by the methods of Example 3, and Comparative Examples 1 to 3 (FIG. 6A) and comparison of sequence specificity of nucleases expressed by these vectors (FIG. 6B).

BEST MODE FOR CARRYING OUT THE INVENTION

In the first aspect, the present invention provides a polypeptide comprising a DNA-binding domain and a function domain. A DNA-binding domain may be derived from TALE (Transcription Activator-Like Effector) of plant pathogen Xanthomonas, Zinc finger and the like.

A function domain includes a domain coding for an enzyme, a transcriptional regulatory element, a reporter protein and the like. The enzyme includes a DNA modification enzyme such as recombinase, nuclease, ligase, kinase, phosphatase; and other enzymes such as lactamase and the like. As used herein, a domain coding for nuclease is referred to as a DNA-cleaving domain. The transcriptional regulatory element includes activator, repressor and the like. The reporter protein includes a fluorescent protein such as green fluorescent protein (GFP), humanized Renilla green fluorescent protein (hrGFP), enhanced green fluorescent protein (eGFP), enhanced blue fluorescent protein (eBFP), enhanced cyan fluorescent protein (eCFP), enhanced yellow fluorescent protein (eYFP), red fluorescent protein (RFP or DsRed), mCherry and the like; a bioluminescent protein such as firefly luciferase, Renilla luciferase and the like; an enzyme converting a chemiluminescent substrate such as alkaline phosphatase, peroxidase, chloramphenicol acetyltransferase, β-galactosidase and the like. A DNA-cleaving domain is preferably the one that is close by another DNA-cleaving domain to form a multimer to obtain an improved nuclease activity. Such a DNA-cleaving domain includes those from FokI and the like.

In the first aspect of the polypeptide of the present invention, a DNA-binding domain and a function domain are linked to each other via a polypeptide consisting of 35-55, preferably 40-50, more preferably 45-49, most preferably 47 amino acid residues. A polypeptide through which a DNA-binding domain and a function domain are linked to each other includes, for instance, a polypeptide consisting of the amino acid sequence of from position 754 to position 801 of SEQ ID NO: 34 as well as a polypeptide having sequence identity of 85%, 90%, 95%, or 97% with the amino acid sequence of from position 754 to position 801 of SEQ ID NO: 34.

In the first aspect of the polypeptide of the present invention, a DNA-binding domain comprises a plurality of DNA-binding modules consecutively from the N-terminus. A single DNA-binding module recognizes specifically a single base pair. The number of a DNA-binding module contained in a DNA-binding domain is preferably 8-40, more preferably 12-25, even more preferably 15-20 in view of compatibility between a high level of function of a function domain and a high level of specificity of DNA sequence recognition. A DNA-binding module includes, for instance, TAL effector repeat and the like. The length of a DNA-binding module includes, for instance, 20-45, 30-38, 32-36, or 34. The length of DNA-binding modules contained in a DNA-binding domain is preferably the same for all the DNA-binding modules. A DNA-binding module includes, for instance, a polypeptide consisting of the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. When the amino acid residues at positions 12 and 13 in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 are H and D, respectively, said DNA-binding domain recognizes C as a nucleotide. When the amino acid residues at positions 12 and 13 are N and G, respectively, said DNA-binding domain recognizes T as a nucleotide. When the amino acid residues at positions 12 and 13 are N and I, respectively, said DNA-binding domain recognizes A as a nucleotide. When the amino acid residues at positions 12 and 13 are N and N, respectively, said DNA-binding domain recognizes G as a nucleotide. A DNA-binding module includes, for instance, a polypeptide which has sequence identity of 85%, 90%, 95%, or 97% with the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 and which substantially retains the function to recognize a single nucleotide.

In the first aspect of the polypeptide of the present invention, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-3 counted from the N-terminus is the same for any of n. A combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-2 counted from the N-terminus is the same for any of n. A combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-1 counted from the N-terminus is the same for any of n. A combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 n counted from the N-terminus is the same for any of n. In this context, n is natural number of 1 to 10, preferably natural number of 1 to 7, more preferably natural number of 1 to 5 and is preferably natural number that is sufficient for referring to all the DNA-binding modules contained in a DNA-binding domain. In this context, x is natural number of 1 to 40, preferably natural number of 1 to 10, more preferably natural number of 2 to 6, even more preferably natural number of 3 to 5, most preferably natural number of 4. In this context, y is natural number of 1 to 40, preferably natural number of 25 to 40, more preferably natural number of 30 to 36, even more preferably natural number of 31 to 33, most preferably natural number of 32. In this context, x and y are different natural number from each other. The values of x and y may vary depending on the length of a DNA-binding module used. In this context, x preferably represents the number indicating the position corresponding to the amino acid residue at position 4 in a DNA-binding module consisting of 34 amino acid residues whereas y preferably represents the number indicating the position corresponding to the amino acid residue at position 32 in a DNA-binding module consisting of 34 amino acid residues.

In the first aspect of the polypeptide of the present invention, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-3 counted from the N-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-2 counted from the N-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-1 counted from the N-terminus, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n counted from the N-terminus are different from each other. In this context, n is natural number of 1 to 10, preferably natural number of 1 to 7, more preferably natural number of 1 to 5 and is preferably natural number that is sufficient for referring to all the DNA-binding modules contained in a DNA-binding domain. In this context, x is natural number of 1 to 40, preferably natural number of 1 to 10, more preferably natural number of 2 to 6, even more preferably natural number of 3 to 5, most preferably natural number of 4. In this context, y is natural number of 1 to 40, preferably natural number of 25 to 40, more preferably natural number of 30 to 36, even more preferably natural number of 31 to 33, most preferably natural number of 32. In this context, x and y are different natural number from each other. Preferably, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-3 counted from the N-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-2 counted from the N-terminus, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n counted from the N-terminus are selected from the group consisting of a combination of D and D, a combination of E and A, a combination of D and A, and a combination of A and D, respectively for x and y in this order.

In the second aspect, the present invention provides the polypeptide of the first aspect wherein the function domain is a DNA-cleaving domain.

In the third aspect, the present invention provides a vector comprising a polynucleotide coding for the polypeptide of the first aspect or the second aspect. A vector includes a plasmid vector, a cosmid vector, a viral vector, an artificial chromosome vector and the like. An artificial chromosome vector includes a yeast artificial chromosome vector (YAC), a bacterial artificial chromosome vector (BAC), a P1 artificial chromosome vector (PAC), a mouse artificial chromosome vector (MAC), and a human artificial chromosome vector (HAC). A component of a vector includes a nucleic acid such as DNA, RNA and the like, a nucleic acid analogue such as GNA, LNA, BNA, PNA, TNA and the like. A vector may be modified with a component other than a nucleic acid such as a saccharide.

By introducing the vector of the third aspect of the present invention into cells and the like for expression, the polypeptide of the first aspect or the second aspect of the present invention can be prepared. Also, by introducing the vector of the third aspect of the present invention into cells and the like for expression, a desired function corresponding to a function domain can be fulfilled in cells such as DNA modification such as DNA recombination, DNA cleavage, etc.; expression of other enzymatic activity such as transcriptional regulation; labelling of a DNA region by a reporter protein. In case that a function domain is a DNA-cleaving domain, by introducing plural, preferably two, of the vectors of the third aspect of the present invention into cells and the like for expression, nucleotide sequence-specific double strand cleavage can be induced on a genomic DNA of the cells where the vector is introduced so that mutation is introduced in the genome of the cells. The source of cells to which the vector of the third aspect of the present invention is introduced includes an animal such as mammal, e.g. Drosophila, zebrafish and mouse, a plant such as Arabidopsis thaliana, culture cells such as ES cells, iPS cells, and the like.

In the fourth aspect, the present invention provides a vector library for preparing the vector of the third aspect. The vector library of the fourth aspect of the present invention is composed of a plurality of vectors. The vector library preferably comprises vectors useful for preparing the vector of the third aspect exhaustively with regard to a combination of four kinds of nucleotide which a combination of four DNA-binding modules recognizes. However, as far as the manufacture of the vector of the third aspect is possible, the vector library may comprise vectors not exhaustively. The vector library of the fourth aspect of the present invention comprises vectors for exhaustively constructing the polypeptide of the first aspect or the second aspect of the present invention comprising e.g. 6 to 9, 10 to 13, 14 to 17, or 18 to 21 DNA-binding modules. The polypeptide of the first aspect or the second aspect of the present invention, when having 14 to 21 DNA-binding modules, is particularly excellent in compatibility between a high level of specificity of sequence recognition and a high level of function of a function domain. Thus, such a vector library, though comprising a rather small number of vectors, is excellent as allowing for the manufacture of the polypeptide of the first aspect or the second aspect with high effects by means of simple procedures.

All the vectors constituting the vector library of the fourth aspect of the present invention comprise a first restriction site, a polynucleotide coding for four DNA-binding modules and a second restriction site in this order from the 5′-end.

A combination of a first restriction site and a second restriction site contained in a vector constituting the vector library of the fourth aspect of the present invention is any one of a combination of a restriction site of type A and a restriction site of type B, a combination of a restriction site of type A and a restriction site of type C, a combination of a restriction site of type A and a restriction site of type D, a combination of a restriction site of type A and a restriction site of type E, a combination of a restriction site of type B and a restriction site of type C, a combination of a restriction site of type C and a restriction site of type D, and a combination of a restriction site of type D and a restriction site of type E. Types A to E indicated in relation to a restriction site are used herein for descriptive purposes for showing difference in property of the respective restriction sites. In case that the types are different from each other, property of their restriction sites is different whereas in case that the types are the same, property of their restriction sites is the same. In the vector library of the fourth aspect of the present invention, the restriction sites of type A to type E are cleaved by the same restriction enzyme. Also, the restriction sites of type A to type E are cleaved by the same restriction enzyme to thereby produce cleaved terminals different from each other. Such a restriction site includes the one by a restriction enzyme that cleaves an arbitrary site adjacent to a recognition site of the restriction enzyme, for instance, BsaI, BbsI, BsmBI and the like.

As shown in FIG. 1, STEP 2, for the manufacture of the vector of the third aspect of the present invention comprising 18 to 21 DNA-binding modules, the vector library of the fourth aspect of the present invention comprises a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type A and a restriction site of type B, a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type B and a restriction site of type C, a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type C and a restriction site of type D, and a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type D and a restriction site of type E, preferably exhaustively with regard to nucleotides recognized by DNA-binding modules.

Also as shown in FIG. 1, STEP 2, for the manufacture of the vector of the third aspect of the present invention comprising 14 to 17 DNA-binding modules, the vector library of the fourth aspect of the present invention comprises a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type A and a restriction site of type C, a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type C and a restriction site of type D, and a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type D and a restriction site of type E, preferably exhaustively with regard to nucleotides recognized by DNA-binding modules.

Besides, as shown in FIG. 1, STEP 2, for the manufacture of the vector of the third aspect of the present invention comprising 10 to 13 DNA-binding modules, the vector library of the fourth aspect of the present invention comprises a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type A and a restriction site of type D, and a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type D and a restriction site of type E, preferably exhaustively with regard to nucleotides recognized by DNA-binding modules.

Furthermore, as shown in FIG. 1, STEP 2, for the manufacture of the vector of the third aspect of the present invention comprising 6 to 9 DNA-binding modules, the vector library of the fourth aspect of the present invention comprises a vector wherein a combination of a first restriction site and a second restriction site is a combination of a restriction site of type A and a restriction site of type E, preferably exhaustively with regard to nucleotides recognized by DNA-binding modules.

The vectors constituting the vector library of the fourth aspect of the present invention comprises DNA-binding modules. The length of a DNA-binding module includes, for instance, 30 to 38, 32 to 36, or 34. The length of DNA-binding modules contained in a DNA-binding domain is preferably the same for all the vectors constituting the vector library. Also, the length of DNA-binding modules contained in a DNA-binding domain is preferably the same for all the four DNA-binding modules contained in the vector constituting the vector library. A DNA-binding module includes, for instance, a polypeptide consisting of the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. A DNA-binding module includes, for instance, a polypeptide which has sequence identity of 85%, 90%, 95%, or 97% with the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 and which substantially retains the function to recognize a single nucleotide.

Among the four DNA-binding modules contained in vectors constituting the vector library of the fourth aspect of the present invention, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 1 counted from the 5′-terminus is the same for an arbitrary vector constituting the vector library. Likewise, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 2 counted from the 5′-terminus is the same for an arbitrary vector constituting the vector library. Also, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 3 counted from the 5′-terminus is the same for an arbitrary vector constituting the vector library. Also, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 counted from the 5′-terminus is the same for an arbitrary vector constituting the vector library. In this context, x is natural number of 1 to 40, preferably natural number of 1 to 10, more preferably natural number of 2 to 6, even more preferably natural number of 3 to 5, most preferably natural number of 4. In this context, y is natural number of 1 to 40, preferably natural number of 25 to 40, more preferably natural number of 30 to 36, even more preferably natural number of 31 to 33, most preferably natural number of 32. In this context, x and y are different natural number from each other. The values of x and y may vary depending on the length of a DNA-binding module used. In this context, x preferably represents the number indicating the position corresponding to the amino acid residue at position 4 in a DNA-binding module consisting of amino acid residues whereas y preferably represents the number indicating the position corresponding to the amino acid residue at position 32 in a DNA-binding module consisting of 34 amino acid residues.

Among the four DNA-binding modules contained in vectors constituting the vector library of the fourth aspect of the present invention, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 1 counted from the 5′-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 2 counted from the 5′-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 3 counted from the 5′-terminus, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 counted from the 5′-terminus, are different from each other. In this context, x is natural number of 1 to 40, preferably natural number of 1 to 10, more preferably natural number of 2 to 6, even more preferably natural number of 3 to 5, most preferably natural number of 4. In this context, y is natural number of 1 to 40, preferably natural number of 25 to 40, more preferably natural number of 30 to 36, even more preferably natural number of 31 to 33, most preferably natural number of 32. In this context, x and y are different natural number from each other. The values of x and y may be for instance those as described above. Preferably, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 1 counted from the 5′-terminus is D and D, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 2 counted from the 5′-terminus is E and A, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 3 counted from the 5′-terminus is D and A, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 counted from the 5′-terminus is A and D, respectively for x and y in this order.

By using the vector library of the fourth aspect of the present invention, the vector of the third aspect of the present invention can be prepared with simple procedures. Specifically, vectors corresponding to the sequence of DNA-binding modules contained in the vector of the third aspect of the present invention are selected from the vector library of the fourth aspect of the present invention, the selected vectors are digested with restriction enzymes that cleave restriction sites of types A to E and the vector fragments obtained by digestion are linked together to prepare the vector of the third aspect of the present invention. All the vectors constituting the vector library of the fourth aspect of the present invention have two restriction sites, which are cleaved by the same restriction enzyme and produce cleaved terminals different from each other as a consequence of cleavage by said enzyme. Thus, for the manufacture of the vector of the third aspect of the present invention, digestion of the selected vectors and ligation of the vector fragments can be performed in one and the same reaction solution, respectively. Therefore, by using the vector library of the fourth aspect of the present invention, the vector of the third aspect of the present invention can be prepared with quite simple procedures.

In the fifth aspect, the present invention provides a vector set for preparing the vector library of the fourth aspect.

The vector set of the fifth aspect of the present invention comprises a plurality of vectors. The vector set preferably comprises vectors useful for preparing the vector library of the fourth aspect exhaustively. However, as far as the manufacture of the vector library of the fourth aspect is possible, the vector set may comprise vectors not exhaustively.

All the vectors contained in the vector set of the fifth aspect of the present invention comprises a first restriction site, a DNA-binding module and a second restriction site in this order from the 5′-end. The first restriction site and the second restriction site are preferably the ones not cleaved by a restriction enzyme that cleaves the first restriction site and the second restriction site contained in the vectors constituting the vector library of the fourth aspect of the present invention. In this regard, the vector of the third aspect can be prepared from the vector library of the fourth aspect with simpler procedures.

The length of a DNA-binding module in the vector contained in the vector set of the fifth aspect of the present invention is preferably the same for all the vectors contained in the vector set. A DNA-binding module includes, for instance, a polypeptide consisting of the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. A DNA-binding module includes, for instance, a polypeptide which has sequence identity of 85%, 90%, 95%, or 97% with the amino acid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 and which substantially retains the function to recognize a single nucleotide.

The first restriction site and the second restriction site in the vector contained in the vector set of the fifth aspect of the present invention are cleaved by the same restriction enzyme. The first restriction site and the second restriction site are also cleaved by the same restriction enzyme to thereby produce cleaved terminals different from each other. Such a restriction site includes the one by a restriction enzyme that cleaves an arbitrary site adjacent to a recognition site of the restriction enzyme, for instance, BsaI, BbsI, BsmBI and the like.

A combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module in the vector contained in the vector set of the fifth aspect of the present invention is any one of four different combinations. The four different combinations of an amino acid residue at position x and an amino acid residue at position y includes, for instance, a combination of D and D, a combination of E and A, a combination of D and A, and a combination of A and D, for x and y in this order. In this context, x is natural number of 1 to 40, preferably natural number of 1 to 10, more preferably natural number of 2 to 6, even more preferably natural number of 3 to 5, most preferably natural number of 4. In this context, y is natural number of 1 to 40, preferably natural number of 25 to 40, more preferably natural number of 30 to 36, even more preferably natural number of 31 to 33, most preferably natural number of 32. In this context, x and y are different natural number from each other. The values of x and y may vary depending on the length of a DNA-binding module used.

In this context, x preferably represents the number indicating the position corresponding to the amino acid residue at position 4 in a DNA-binding module consisting of amino acid residues whereas y preferably represents the number indicating the position corresponding to the amino acid residue at position 32 in a DNA-binding module consisting of 34 amino acid residues.

The vectors contained in the vector set of the fifth aspect of the present invention include a vector in which a combination of the first restriction site and the second restriction site is a combination of a type a restriction site and a type β restriction site and a DNA-binding module recognizes nucleotide of A, T, G, or C; a vector in which a combination of the first restriction site and the second restriction site is a combination of a type β restriction site and a type y restriction site and a DNA-binding module recognizes nucleotide of A, T, G, or C; a vector in which a combination of the first restriction site and the second restriction site is a combination of a type y restriction site and a type 5 restriction site and a DNA-binding module recognizes nucleotide of A, T, G, or C; a vector in which a combination of the first restriction site and the second restriction site is a combination of a type 5 restriction site and a type restriction site and a DNA-binding module recognizes nucleotide of A, T, G, or C. In this context, types a to 5 of the restriction site as used herein are expediently set for showing difference in property of the restriction site, denoting that different types of the restriction site are different in their property from each other whereas the same types of the restriction site are common in their property. The vector set of the fifth aspect of the present invention preferably include all the vectors mentioned above. In this case, every mode of the vector library of the fourth aspect of the present invention can be prepared.

By using the vector set of the fifth aspect of the present invention, the vector library of the fourth aspect of the present invention can be prepared with simple procedures. Specifically, four vectors are selected from the vector set of the fifth aspect of the present invention based on a combination of the four DNA-binding modules contained in vectors constituting the vector library of the fourth aspect of the present invention, the selected vectors are digested with a restriction enzyme that cleaves the first restriction site and the second restriction site and the vector fragments obtained by digestion are linked together to prepare the vector library of the fourth aspect of the present invention. All the vectors contained in the vector set of the fifth aspect of the present invention have two restriction sites, which are cleaved by the same restriction enzyme and produce cleaved terminals different from each other as a consequence of cleavage by said enzyme. Thus, for the manufacture of the vector library of the fourth aspect of the present invention, digestion of the selected vectors and ligation of the vector fragments can be performed in one and the same reaction solution, respectively. Therefore, by using the vector set of the fifth aspect of the present invention, the vector library of the fourth aspect of the present invention can be prepared with quite simple procedures.

EXAMPLE 1

The present invention is further explained in more detail by means of the following Examples but is not limited thereto.

EXAMPLE 1 Preparation of Vector Set

The nucleotide sequence shown in FIG. 2 with addition at both ends of the recognition site of restriction enzyme BsaI was prepared by artificial gene synthesis and inserted into pBluescript SK vector to prepare a vector set (p1HD-p4HD, p1NG-p4NG, p1NI-p4NI, p1NN-p4NN) for use in STEP 1 of FIG. 1.

EXAMPLE 2 Preparation of Vector Library

Using pFUS B6 vector (Addgene) as a template, pFUS2 vector shown in STEP 1 of FIG. 1 was prepared by In-Fusion cloning (Clontech). As shown in STEP 1 of FIG. 1, using the prepared pFUS2 vector and the vector set prepared in Example 1, the Golden Gate reaction was performed to prepare a vector library.

EXAMPLE 3 Preparation of TALEN Expression Vector

Using pTALEN_v2 and pcDNA-TAL-NC2 (both Addgene), In-Fusion cloning was performed, a BsmBI site adjacent sequence for incorporating modules was prepared by In-Fusion cloning, and a globin leader sequence was introduced upstream the initiation codon by In-Fusion cloning to prepare ptCMV vectors as shown in FIG. 1, STEP 2. Using the prepared ptCMV vectors, the vector library prepared in Example 2, and the vectors contained in Golden Gate TALEN and TAL Effector Kit, Yamamoto Lab TALEN Accessory Pack (both Addgene), DNA-binding domains were inserted by Golden Gate procedure as shown in FIG. 1, STEP 2 to prepare TALEN expression vectors. The ptCMV vector was used in which the number of amino acid residues of a region adjacent to the N-terminus of DNA-binding domain (TALEN-N′) is 153 and the number of amino acid residues of a region franked by the C-terminus of DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is 47. FIG. 3 shows the nucleotide and amino acid sequences of an example of TALEN prepared.

As shown in FIG. 3, the amino acid residues at position 4 and at position 32 of DNA-binding modules (34 amino acid residues in total) in the TALEN expression vector of Example 3 were different from each other among DNA-binding modules at position 4n-3, at position 4n-2, at position 4n-1 and at position 4n (n is natural number). The amino acid residues at position 4 and at position 32 in DNA-binding modules at position 4n-3 (n is natural number) were common among the respective DNA-binding modules. The same was applied to DNA-binding modules at position 4n-3, at position 4n-2, at position 4n-1 and at position 4n (n is natural number). As such, by using the vector set of Example 1 and the vector library of Example 2, the TALEN expression vectors could be prepared with a repetitive fashion for every four DNA-binding modules.

COMPARATIVE EXAMPLE 1 Preparation of TALEN Expression Vector

The Golden Gate reaction was performed as described in Example 2 except that pHD1-6, pNG1-6, pNI1-6, pNN1-6 contained in Golden Gate TALEN and TAL Effector Kit (Addgene) was used in place of the vector set prepared in Example 1 and that Yamamoto Lab TALEN Accessory Pack (Addgene) was used as pFUS vector for use in the reaction, to prepare a vector library. Using the prepared vector library, the Golden Gate reaction was performed as described in Example 3 to prepare the TALEN expression vectors.

COMPARATIVE EXAMPLE 2 Preparation of TALEN Expression Vector

The procedures of Example 3 were carried out except that the ptCMV vector was used in which the number of amino acid residues of a region adjacent to the N-terminus of DNA-binding domain (TALEN-N′) is 136 and the number of amino acid residues of a region franked by the C-terminus of DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is 63, to prepare TALEN expression vectors.

COMPARATIVE EXAMPLE 3 Preparation of TALEN Expression Vector

The procedures of Comparative Example 1 were carried out except that the ptCMV vector was used in which the number of amino acid residues of a region adjacent to the N-terminus of DNA-binding domain (TALEN-N′) is 136 and the number of amino acid residues of a region franked by the C-terminus of DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is 63, to prepare TALEN expression vectors.

TEST EXAMPLE 1 Assessment of Recognition Specificity of TALEN

The TALEN expression vectors (L14 to L20 and R14 to R20) recognizing the sites indicated in FIG. 4B were prepared as in Example 3 or in Comparative Examples 1 to 3. Each one from L14 to L20 and from R14 to R20 as prepared were combined together as shown in FIG. 4C to give right and left TALEN expression vectors. Using the sequences shown in FIG. 4B as a target sequence for TALEN, Single Strand Annealing Assay (cf. Non-patent reference 5) was conducted with HEK293T cell to assess the TALEN activity.

Specifically, Single Strand Annealing Assay was performed as described below. First, a reporter vector was prepared in which a target sequence of TALEN of interest was inserted into a reporter vector (pGL4-SSA; Addgene) wherein a segmented firefly luciferase gene was linked downstream CMV promoter. The target sequence of TALEN was prepared by annealing synthetic oligonucleotides and was inserted into pGL4-SSA vector treated with BsaI using Ligation-Convenience Kit (NIPPON GENE CO., LTD.). Then, the prepared reporter vector together with the TALEN expression vector and pRL-CMV vector (Promega), which is an expression vector of Renilla luciferase, were introduced into HEK293T cells on 96-well plate by lipofectin procedure. After culture for 24 hours, the reporter activity was measured using Dual-Glo Luciferase Assay System (Promega). An amount of DNA introduced is each 200 ng for the right and left TALEN expression vectors, 100 ng for the reporter vector, and 20 ng for the pRL-CMV vector. Measurement of chemoluminescence was done with TriStar LB 941 plate reader (Berthold Japan K.K.).

FIG. 4C shows relative values of the reporter activity for the respective combinations of the right and left TALEN expression vectors in Example 3 and Comparative Examples 1 to 3 in comparison with a combination of L20 and R17 in Comparative Example 1. Table of FIG. 4B shows the length (the number of nucleotides) of the spacer region franked by the right and left TALEN recognition sites for the respective combinations of the right and left TALEN expression vectors.

As shown in FIG. 4C, in case of Example 3 and Comparative Example 1, a specifically higher activity was obtained only for the limited cases of 12 to 15 of the length of the spacer region, demonstrating that specific cleavage is possible for the limited spacer region. On the other hand, in case of Comparative Example 2 and Comparative Example 3, the level of the activity has no relevance with the length of the spacer region, demonstrating that the possibility is high that sequences with different length of the spacer region are recognized and cleaved. It was thus demonstrated that, by using the TALEN expression vectors of Example 3, efficient DNA cleavage can be conducted with less non-specific cleavage of sequences with different length of the spacer region.

TEST EXAMPLE 2 Assessment of Activity of TALEN

A pair of the right and left TALEN expression vectors that recognize the site shown in FIG. 5A (ATM (L17 for the left, R17 for the right), APC (L17 for the left, R17 for the right) and eGFP (L20 for the left, R18 for the right)) were prepared as in Example 3 or Comparative Examples 1 to 3. Using a pair of the right and left prepared by the respective procedures as the right and left TALEN expression vectors, Single Strand Annealing Assay (Non-patent reference 5) was performed with HEK293T cells as in Test Example 1 using the sequence shown in FIG. 5A as a targeting sequence of TALEN to assess the activity of TALEN.

The results are shown in FIG. 5B in which the axis of ordinate indicates relative values of the reporter activity in comparison with a combination of L20 and R17 of Comparative Example 1 prepared in Test Example 1. As shown in FIG. 5B, in case of Example 3, a higher DNA cleavage activity of TALEN was observed for any of ATM, APC and eGPF as compared to Comparative Example 1. This proved that the repetitive structure of the DNA-binding modules in accordance with the present invention renders the DNA cleavage activity of TALEN be improved.

TEST EXAMPLE 3 Assessment of Recognition Specificity of TALEN

A pair of the right and left TALEN expression vectors that recognize the site shown in FIG. 6A (L19 for the left, R18 for the right) were prepared as in Example 3 or Comparative Examples 1 to 3. Using a pair of the right and left prepared by the respective procedures as the right and left TALEN expression vectors, Single Strand Annealing Assay (Non-patent reference 5) was performed with HEK293T cells as in Test Example 1 using the sequence shown in FIG. 6A (no mismatches, 1 left mismatch and 0 right mismatch (L:1 mismatch/R:0 mismatch), 1 left mismatch and 1 right mismatch (L:1 mismatch/R:1 mismatch), or 2 left mismatches and 2 right mismatches (L:2 mismatches/R:2 mismatches)) as a targeting sequence of TALEN to assess the activity of TALEN. In case that the respective sequences shown in FIG. 6A are used as a targeting sequence of TALEN, mismatch occurs at lower cases in FIG. 6A and thus the level of recognition specificity of TALEN used can be compared by comparing the results of TALEN activity assessment of the targeting sequence of TALEN used.

The results are shown in FIG. 6B in which the axis of ordinate indicates relative values of the measurement of the firefly luciferase activity divided by the measurement of the Renilla luciferase activity. As shown in FIG. 6B, in case that the TALEN expression vectors of Comparative Example were used, even in case of the sequence of 2 left mismatches and 2 right mismatches, a high activity was observed and thus the recognition specificity of the TALEN expression vectors of Comparative Example 2 was low. On the other hand, in case that the TALEN expression vectors of Example 3 were used, in case of the sequence of 2 left mismatches and 2 right mismatches, almost complete loss of the activity was observed. This proved that the TALEN expression vectors of Example 3 can afford to DNA cleavage with high specificity while maintaining a high cleavage activity as shown in Comparative Example 2. Therefore, it was found that a target DNA of interest can be cleaved safely with high probability by using the TALEN expression vectors of Example 3.

INDUSTRIAL APPLICABILITY

The present invention is useful e.g. for production of a variety of substance by genetic engineering technique and can widely be used in the field of medicine, engineering and agriculture. 

1-5. (canceled)
 6. A Transcription activator-like effector nuclease (TALEN) polypeptide comprising a DNA-binding domain and a DNA-cleaving domain, wherein the DNA-binding domain and the DNA-cleaving domain are linked to each other via a polypeptide consisting of 40 to 50 amino acid residues, wherein the DNA-binding domain comprises 16 to 20 of DNA-binding modules consisting of 34 amino acid residues consecutively from the N-terminus, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-3 counted from the N-terminus is the same for any of n, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-2 counted from the N-terminus is the same for any of n, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-1 counted from the N-terminus is the same for any of n, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n counted from the N-terminus is the same for any of n, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-3 counted from the N-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-2 counted from the N-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n-1 counted from the N-terminus, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4n counted from the N-terminus are different from each other, and wherein n is natural number of 1 to 10, x is natural number of 1 to 40, y is natural number of 1 to 40, and x and y are different natural number from each other.
 7. A vector comprising a polynucleotide coding for the TALEN polypeptide as set forth in claim 6 wherein the number of amino acid residues of a region adjacent to the N-terminus of DNA-binding domain (TALEN-N′) is 153 and the number of amino acid residues of a region franked by the C-terminus of DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is
 47. 8. A vector library for preparing the vector as set forth in claim 7, wherein the vector library consists of a plurality of vectors, each of which vector has a first restriction site, a polynucleotide coding for four DNA-binding modules and a second restriction site in this order from the 5′-end, wherein a combination of the first restriction site and the second restriction site is any one of a combination of a restriction site of type A and a restriction site of type B, a combination of a restriction site of type A and a restriction site of type C, a combination of a restriction site of type A and a restriction site of type D, a combination of a restriction site of type A and a restriction site of type E, a combination of a restriction site of type B and a restriction site of type C, a combination of a restriction site of type C and a restriction site of type D, and a combination of a restriction site of type D and a restriction site of type E, wherein each of the restriction sites of types A to E produce different cleaved terminals when cleaved with the same restriction enzyme; and among the four DNA-binding modules, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 1 counted from the 5′-terminus is the same for any of the vectors, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 2 counted from the 5′-terminus is the same for any of the vectors, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 3 counted from the 5′-terminus is the same for any of the vectors, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 counted from the 5′-terminus is the same for any of the vectors, and wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 1 counted from the 5′-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 2 counted from the 5′-terminus, a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 3 counted from the 5′-terminus, and a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module at position 4 counted from the 5′-terminus are different from each other, and wherein x is natural number of 1 to 40, y is natural number of 1 to 40, and x and y are different natural number from each other.
 9. A vector set for preparing the vector library as set forth in claim 8, wherein the vector set comprises a plurality of vectors, each of which vector comprises a first restriction site, a DNA-binding module and a second restriction site in this order from the 5′-end, wherein the first restriction site and the second restriction site produce different cleaved terminals when cleaved with the same restriction enzyme, wherein the first restriction site and the second restriction site are the ones not cleaved by a restriction enzyme that cleaves the first restriction site and the second restriction site contained in the vectors constituting the vector library as set forth in claim 8, wherein a combination of an amino acid residue at position x and an amino acid residue at position y in a DNA-binding module is any of the four different combinations, and wherein x is natural number of 1 to 40, y is natural number of 1 to 40, and x and y are different natural number from each other. 